Publisher Correction: SLX4 interacts with RTEL1 to prevent transcription-mediated DNA replication perturbations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: SLX4 interacts with RTEL1 to prevent transcription-mediated DNA replication perturbations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

SLX4 interacts with RTEL1 to prevent transcription-mediated DNA replication perturbations.

The SLX4 tumor suppressor is a scaffold that plays a pivotal role in several aspects of genome protection, including homologous recombination, interstrand DNA crosslink repair and the maintenance of common fragile sites and telomeres. Here, we unravel an unexpected direct interaction between SLX4 and the DNA helicase RTEL1, which, until now, were viewed as having independent and antagonistic functions. We identify cancer and Hoyeraal-Hreidarsson syndrome-associated mutations in SLX4 and RTEL1, respectively, that abolish SLX4-RTEL1 complex formation. We show that both proteins are recruited to nascent DNA, tightly co-localize with active RNA pol II, and that SLX4, in complex with RTEL1, promotes FANCD2/RNA pol II co-localization. Importantly, disrupting the SLX4-RTEL1 interaction leads to DNA replication defects in unstressed cells, which are rescued by inhibiting transcription. Our data demonstrate that SLX4 and RTEL1 interact to prevent replication-transcription conflicts and provide evidence that this is independent of the nuclease scaffold function of SLX4.

Cancer cell-derived long pentraxin 3 (PTX3) promotes melanoma migration through a toll-like receptor 4 (TLR4)/NF-κB signaling pathway.

Cutaneous melanoma is one of the most aggressive cancers characterized by a high plasticity, a propensity for metastasis, and drug resistance. Melanomas are composed of phenotypically diverse subpopulations of tumor cells with heterogeneous molecular profiles that reflect intrinsic invasive abilities. In an attempt to identify novel factors of the melanoma invasive cell state, we previously investigated the nature of the invasive secretome by using a comparative proteomic approach. Here, we have extended this analysis to show that PTX3, an acute phase inflammatory glycoprotein, is one such factor secreted by invasive melanoma to promote tumor cell invasiveness. Elevated PTX3 production was observed in the population of MITFlow invasive cells but not in the population of MITFhigh differentiated melanoma cells. Consistently, MITF knockdown increased PTX3 expression in MITFhigh proliferative and poorly invasive cells. High levels of PTX3 were found in tissues and blood of metastatic melanoma patients, and in BRAF inhibitor-resistant melanoma cells displaying a mesenchymal invasive MITFlow phenotype. Genetic silencing of PTX3 in invasive melanoma cells dramatically impaired migration and invasion in vitro and in experimental lung extravasation assay in xenografted mice. In contrast, addition of melanoma-derived or recombinant PTX3, or expression of PTX3 enhanced motility of low migratory cells. Mechanistically, autocrine production of PTX3 by melanoma cells triggered an IKK/NFκB signaling pathway that promotes migration, invasion, and expression of the EMT factor TWIST1. Finally, we found that TLR4 and MYD88 knockdown inhibited PTX3-induced melanoma cell migration, suggesting that PTX3 functions through a TLR4-dependent pathway. Our work reveals that tumor-derived PTX3 contributes to melanoma cell invasion via targetable inflammation-related pathways. In addition to providing new insights into the biology of melanoma invasive behavior, this study underscores the notion that secreted PTX3 represents a potential biomarker and therapeutic target in a subpopulation of MITFlow invasive and/or refractory melanoma.

Septin 9_i2 is downregulated in tumors, impairs cancer cell migration and alters subnuclear actin filaments.

Functions of septin cytoskeletal polymers in tumorigenesis are still poorly defined. Their role in the regulation of cytokinesis and cell migration were proposed to contribute to cancer associated aneuploidy and metastasis. Overexpression of Septin 9 (Sept9) promotes migration of cancer cell lines. SEPT9 mRNA and protein expression is increased in breast tumors compared to normal and peritumoral tissues and amplification of SEPT9 gene was positively correlated with breast tumor progression. However, the existence of multiple isoforms of Sept9 is a confounding factor in the analysis of Sept9 functions. In the present study, we analyze the protein expression of Sept9_i2, an uncharacterized isoform, in breast cancer cell lines and tumors and describe its specific impact on cancer cell migration and Sept9 cytoskeletal distribution. Collectively, our results showed that, contrary to Sept9_i1, Sept9_i2 did not support cancer cell migration, and induced a loss of subnuclear actin filaments. These effects were dependent on Sept9_i2 specific N-terminal sequence. Sept9_i2 was strongly down-regulated in breast tumors compared to normal mammary tissues. Thus our data indicate that Sept9_i2 is a negative regulator of breast tumorigenesis. We propose that Sept9 tumorigenic properties depend on the balance between Sept9_i1 and Sept9_i2 expression levels.

KIT-D816V oncogenic activity is controlled by the juxtamembrane docking site Y568-Y570.

Mutation of KIT receptor tyrosine kinase at residue D816 results in ligand-independent constitutive kinase activity. This mutation occurs in most patients with mastocytosis, a myeloproliferative neoplasm, and is detected at lower frequencies in acute myeloid leukemia and in germ cell tumors. Other KIT mutations occur in gastrointestinal stromal tumors (GIST) and mucosal melanoma. KIT is considered as a bona fide therapeutic target as c-kit mutations are driving oncogenes in these pathologies. However, several evidences suggest that KIT-D816V mutant is not as aggressive as other KIT mutants. Here, we show that an intracellular docking site in the juxtamembrane region of KIT maintains a negative regulation on KIT-D816V transforming potential. Sixteen signaling proteins were shown to interact with this motif. We further demonstrate that mutation of this site results in signaling modifications, altered gene expression profile and increased transforming activity of KIT-D816V mutant. This result was unexpected as mutations of the homologous sites on wild-type (WT) KIT, or on the related oncogenic FLT3-ITD receptor, impair their function. Our results support the hypothesis that, KIT-D816V mutation is a mild oncogenic event that is sufficient to confer partial transforming properties, but requires additional mutations to acquire its full transforming potential.

Reproducibility and reliability of subscapularis tendon assessment using CT-arthrography.

BACKGROUND: Computed tomography (CT) coupled with arthrography remains the reference standard for the preoperative evaluation of rotator cuff tears. The objectives of this study were to evaluate intra-observer and inter-observer reproducibilities of CT-arthrographic assessment of the subscapularis tendon and to assess the validity and reliability of this investigation. HYPOTHESIS: CT-arthrography is reliable and reproducible for the preoperative characterisation of subscapularis tendon lesions. MATERIAL AND METHODS: We retrospectively reviewed 67 shoulders with rotator cuff tears in one or more tendons managed by arthroscopy, both to confirm the diagnosis and to allow therapeutic interventions. Each of the 67 preoperative CT-arthrograms was evaluated by three readers, of whom the first two evaluated the images twice at an interval of 30 days. The following were recorded at each reading: partial- or full-thickness tear in one or more rotator cuff tendons, intra-tendinous delamination, tendon stump retraction, and fatty degeneration of the muscles. The position of the long head of biceps tendon was assessed. A statistical analysis was performed using Fleiss' method to compute intra-observer and inter-observer variabilities in CT-arthrography assessment of the subscapularis tendon status. Validity of this assessment was measured by computing the concordance coefficients between CT-arthrography and arthroscopy. RESULTS: Specificity of CT-arthrography was satisfactory for assessing the subscapularis tendon. Sensitivity was low. Significant inter-observer and intra-observer variabilities were documented. The concordance coefficients between CT-arthrography and arthroscopy indicated that major differences were common with all three readers. When assessing the subscapularis tendon by CT-arthrography, all readers experienced difficulties in distinguishing intact tendons, delaminated tendons, and tendons with tears confined to the upper third. DISCUSSION: Although CT-arthrography remains the reference standard for the preoperative investigation of rotator cuff tears, significant variability occurs in assessing the continuity of the subscapularis tendon. Reliability of this assessment is not optimal, as shown by our evaluation of concordance with arthroscopy. Although our data should be interpreted in the light of the investigation and measurement biases present in our study, they suggest that CT-arthrography may fail to provide a valid and reproducible assessment of the subscapularis tendon. The development of magnetic resonance (MR) imaging and MR-arthrography will probably improve the preoperative evaluation of subscapularis tears in the near future. LEVEL OF EVIDENCE: Level III; diagnostic value study.

Arthroscopic repair of subscapularis tears: preliminary data from a prospective multicentre study.

BACKGROUND: Until the introduction of arthroscopic-assisted surgery for rotator cuff repair, the frequency of subscapularis tears was underestimated. These tears remain challenging to treat even with arthroscopy. The absence of a specific classification system has hampered communication about the treatment and outcomes of the various types of subscapularis tears. The objective of this prospective multicentre study was to validate the relevance of arthroscopic subscapularis tendon repair based on an assessment of short-term outcomes according to the initial extent of the anatomic lesions. METHODS: A prospective multicentre study sponsored by the French Society for Arthroscopy was conducted from March 2010 to January 2011 in 208 patients with subscapularis lesions that were either isolated or associated with limited anterosuperior tears. The Constant and UCLA scores were used to assess clinical outcomes. Anatomic and prognostic results were evaluated based on the physical examination, preoperative and postoperative imaging study findings, and anatomic lesions. Clinical data were available for 103 patients after at least 1 year of follow-up and radiological data for 129 patients after at least 6 months. RESULTS: The preliminary clinical results in 103 patients with at least 1 year of follow-up showed overall statistically significant improvements in the Constant and UCLA scores, with resolution of the clinical manifestations. The degree of improvement seemed to increase over time. The clinical results varied significantly across patient groups based on a classification system distinguishing four lesion types. Postoperative imaging studies to assess the anatomic results in all patients with at least 6 months of follow-up (n=129) showed tendon healing in 92% of cases but also indicated muscle wasting of the upper subscapularis muscle in 18.6% of cases and increased fatty degeneration of the muscle belly. DISCUSSION: Our study confirms the good clinical and radiological results reported in the literature. Our classification system distinguishing four lesion patterns was applicable during the imaging workup. The main finding from this classification system was the difference in results between Type 2 and Type 3 lesions. The trend towards improvements over time requires confirmation by longer-term studies, which will also have to establish that the increased wasting of the upper subscapularis muscle and fatty degeneration of the muscle belly have no adverse effects.

Diagnosis of subscapularis tendon tears: are available diagnostic tests pertinent for a positive diagnosis?

HYPOTHESIS: Clinically, subscapularis tendon tears are suggested by the presence of increased passive external rotation compared to the opposite side, resisted internal rotation manoeuvres (Lift-Off test [LOT], Belly-Press test [BPT], Napoleon test and Bear-Hug test [BHT] and positive Internal Rotation Lag Sign and/or Belly-Off Signs). Associated bicipital involvement is frequent with subscapularis tendon tears, because it participates in the formation of the biceps pulley. The Palm-Up test (PUT) is used for the biceps, and the Jobe test for the supraspinatus. MATERIAL AND METHODS: In this multicenter study, we evaluated the positive diagnostic value of the clinical tests, LOT, BPT, BHT, PUT, and the Jobe test for subscapularis tears as well as their anatomical value. The relationships of the different parameters studied were compared statistically by analysis of variance (ANOVA). This prospective multicenter study was performed from January 2009 to February 2010 and included 208 cases of subscapularis tendon tears, isolated or associated with partial (Ellman 1, 2 or 3) or full thickness (SFA stage 1) supraspinatus tears. RESULTS: The severity of the subscapularis tear was quantified according to the SFA classification into four stages and according to the level of injury (the lower 1/3 and upper 2/3). The three tests LOT, BPT and BHT were correlated to the severity of observed tears (P<0.05). The more deficient the test results were, the more severe the anatomical damage. The LOT is the test that cannot be performed most often (18%) but when it is positive, it is predictive of very severe tears. The BHT is the most sensitive of all tests (82%). The frequency of biceps involvement was correlated to the severity of subscapularis damage. There was no significant correlation between biceps involvement and subscapularis tests, or between supraspinatus involvement and subscapularis tests. There was no correlation between the Palm-Up test and subscapularis tears with associated supraspinatus involvement however, it was significantly correlated to biceps involvement (P<0.05). The Jobe test was disappointing because it was often positive even for isolated subscapularis tears. CONCLUSION: Even though all three tests were performed (LOT, BPT, BHT), 24% of the subscapularis tears were only diagnosed during surgery. The role of the Internal Rotation Lag Sign and Belly-Off Sign in improving the diagnosis of tears was not studied in this work.

New endoscopic classification for subscapularis lesions.

BACKGROUND: The absence of a coherent classification system has hampered communication about the treatment and outcomes of the various types of subscapularis tendon lesions. In addition, a reliable classification system allows comparisons of epidemiological and therapeutic data. The classification systems used until now fail to incorporate the radiological and intraoperative abnormalities of the bicipital sling, and they do not consider the degree of subscapularis tendon cleavage. Here, we describe a new arthroscopy-based classification system intended for therapeutic and prognostic purposes. METHODS: A prospective multicentre study sponsored by the French Society for Arthroscopy was conducted from March 2010 to January 2011 in 150 isolated subscapularis lesions with or without limited anterosuperior involvement. The bicipital sling and insertion of the deep subscapularis layer were routinely investigated by arthroscopy with video recording. Each lesion was classified after a consensus was reached among four surgeons. RESULTS: We identified four lesion types based on the bicipital sling findings. Type I was defined as partial separation of the subscapularis tendon fibres from the lesser tuberosity with a normal bicipital sling. Type II consisted of a partial subscapularis tear at the lesser tuberosity attachment combined with partial injury to the anterior wall of the bicipital sling, without injury to the superior glenohumeral ligament. Type III was complete separation of the subscapularis fibres from the lesser tuberosity with extensive cleavage of the bicipital sling. Finally, in Type IV, all the subscapularis fibres were detached and, in some cases, conjunction of the subscapularis and supraspinatus fibres produced the comma sign. Nearly all the lesions identified intraoperatively during the study fit one of these four types. DISCUSSION: A reproducible classification system that allows different surgeons to establish comparable homogeneous patient groups is useful for both therapeutic and prognostic purposes. We defined four types of subscapularis lesions that are easy to identify as either isolated lesions or combined with anterosuperior rotator cuff tears. Long head of biceps tendon abnormalities and fatty degeneration of the shoulder muscles can be added to our classification system. Studies of intraobserver and interobserver reproducibility are needed to complete the process of validating the diagnostic and/or prognostic usefulness of this new classification scheme.

Can we improve the indication for Bankart arthroscopic repair? A preliminary clinical study using the ISIS score.

The objectives of this study on arthroscopic treatment of chronic anterior shoulder instability were the collection of the current practices for this indication, their development as reported in the literature, and the analysis of preliminary results on a multicenter prospective series of Bankart arthroscopic procedures undertaken using a common technique on patients selected based on the Instability Severity Index Score (ISIS). This procedure predominates in the English-speaking world, whereas the Latarjet protocol is preferred in France. The choice between the two seems to be cultural since neither technique could be demonstrated to be superior in an analysis of 171 responses to an Internet questionnaire in this study. The literature reports disappointing results in the Bankart arthroscopic procedure and recent articles have researched the predictive factors for its failure. Eleven centers prospectively included 125 patients from 1 December 2007 to 30 November 2008. The inclusion criteria were recurrence of anterior instability and an ISIS less than or equal to four points out of 10. All the selected patients underwent capsuloligamentous reinsertion with a common minimal technique of at least three anchors and four sutures with the same postoperative protocol. At a mean follow-up of 18 months, four patients (3.2%) had experienced recurrence. For the 84 patients reexamined at 1 year, the Walch-Duplay and Rowe scores were, respectively, 88.4 and 87.8 points out of 100. Subjectively, 88.1% of the patients declared they were satisfied and would undergo the intervention again. This study confirmed the use of the ISIS as a consultation tool. Only continuation of the study with a minimum follow-up of 3 years will allow us to validate the lower limit of the ISIS below which this technique could be proposed provided that it respects the technical prerequisite of at least four capsuloligamentous sutures.

Alternative splicing modulates autoinhibition and SH3 accessibility in the Src kinase Fyn.

Src family kinases are central regulators of a large number of signaling pathways. To adapt to the idiosyncrasies of different cell types, these kinases may need a fine-tuning of their intrinsic molecular control mechanisms. Here, we describe on a molecular level how the Fyn kinase uses alternative splicing to adapt to different cellular environments. Using structural analysis, site-directed mutagenesis, and functional analysis, we show how the inclusion of either exon 7A or 7B affects the autoinhibition of Fyn and how this changes the SH3-dependent interaction and tyrosine phosphorylation of Sam68, with functional consequences for the Sam68-regulated survival of epithelial cells. Our results illustrate a novel mechanism of evolution that may contribute to the complexity of Src kinase regulation.

Materno-fetal transmission of human coronaviruses: a prospective pilot study.

This prospective pilot study investigates the possibility of materno-fetal transmission of human coronaviruses (HCoV) responsible for cases of neonatal infection. This vertical transmission was studied with 159 samples from mother-child couples: maternal vaginal (MV) and respiratory (MR) samples during labor; and newborn gastric sample (NG) with detection of HCoV (229E, OC-43, NL-63, HKU1) via real time RT PCR. HCoV was detected in 12 samples (229E: 11; HKU1: 1) from seven mother-child couples. For three couples, only MR tested positive (cases 1-3). For two other couples all three samples (MV, MR and NG) tested positive (cases 4 and 5). For case 6, only MV and NG tested positive. In case 7, only MV was positive. Possible vertical transmission of HCoV was hypothesized in this pilot study and requires further investigation on a larger scale.

[Vertical transmission of human coronavirus. Prospective pilot study]. / Transmission maternofoetale des coronavirus humains. Etude prospective pilote.

UNLABELLED: Human coronaviruses (HCoV) have been implicated in neonatal nosocomial respiratory infection. Prior to our study, several cases of neonatal infection were observed in infants born at our hospital. This prospective pilot monocentric pilot study investigates the possibility of maternofetal transmission of HCoV responsible for cases of neonatal infection observed within the first 24 hours of life. MATERIALS AND METHODS: Three samples from mother-child couples, maternal vaginal (VM) and respiratory (RM) samples during labor; newborn gastric sample (GNN), were assessed for viral analysis using real time RT-PCR for the detection of HCoV 229-E and OC43. Clinical follow-up of infants and mothers was up to Day 3 after birth. RESULTS: One hundred (and) fifty-nine mother-child couples were included between July 2003 and August 2005. HCoV 229-E only was detected in 11 samples from 6 mother-child couples. For 2 couples, all 3 samples (VM, RM and GNN) were tested positive (cases 1 and 2). For case 3, both VM and GNN were positive. For 2 couples, only RM was positive (cases 4 and 5). In case 6, only VM was positive. Of the 3 positive GNN, no infant was symptomatic. CONCLUSION: Possible vertical transmission of HCoV was evidenced in this pilot study and requires further investigation on a larger scale. Equally indicated is the inclusion of tests to detect recently identified human coronaviruses HCoV NL63 and HKU1, as well as genomic profile analysis of HCoV 229-E detected in the 3 positive mother-child couples.

[Clinical results of arthroscopic tenotomy of the long head of the biceps brachii in full thickness tears of the rotator cuff without repair: 40 cases]. / Résultats de la ténotomie arthroscopique du chef long du biceps brachial dans les ruptures transfixiantes de la coiffe des rotateurs non réparées: a propos de 40 cas.

PURPOSE OF THE STUDY: Appropriate treatment of irreparable rotator cuff tears in patients without osteoarthritic shoulder joints remains a subject of debate. Medical treatment, a substitution muscle flap, and palliative arthroscopic treatment have been proposed. Arthroscopic tenotomy of the long head of the biceps brachii is warranted because this tendon is often the cause of part or all of the pain. If there is a full thickness tear of the rotator cuff, the exposed tendon of the long head of the biceps brachii can, because of its anterosuperior position, become impinged against the acromial vault during forward flexion. The purpose of this work was to evaluate the mid-term clinical and radiological results of arthroscopic tenotomy of the long head of the biceps brachii during treatment of full thickness tears of the rotator cuff. MATERIAL AND METHODS: The series included 40 shoulders operated on for tenotomy alone (n=32) or in combination with acromioplasty (n=8). The long head of the biceps brachii was in place in 23 shoulders (58%), displaced in seven and subluxed in five. The position was not determined in five. At last follow-up, the mean rough Constant score was 58 points, giving a gain of 20 points. The gain for pain was +7.1 points, +6.4 points for activity, and +6.6 points for motion. After the operation, muscle force for elbow flexion-supination was decreased 40% compared with an age-, sex- and dominance-matched control group. 86% of the patients were satisfied with the outcome and only two patients were disappointed by the asymmetry of arm muscle volume. Radiographically, at last follow-up there were no signs of superior excentration of the humeral head and the subacromial space, which measured 7.38 mm preoperatively was 7.19 mm postoperatively. Likewise only two shoulders progressed to excentered osteoarthritis at 41 and 72 months. DISCUSSION: Mid-term results of arthroscopic tenotomy of the long head of the biceps brachii are satisfactory. The technique is simple and has limited functional consequences. The procedure has an undeniable impact on pain and has allowed a 34 degree gain in anterior flexion of the shoulder. Complementary acromioplasty was not found to provide a supplementary benefit in this series. Nevertheless, the degradation of the result in one female patient at six years suggests we should be prudent concerning the long-term benefit of this procedure which should be reserved for irreparable tears in patients with minimal functional demands.

[Correction of acquired metatarsus elevatus and hallux flexus: technique used in nine cases]. / Technique de correction du metatarsus elevatus acquis avec hallux flexus. A propos de 9 cas.

Nine cases of acquired metatarus elevatus or horizontalization of the first metatarsal with hallux flexus (dorsal bunion) were treated surgically associating: plantar wedge resection of the base of the first metatarsal or the first cuneiform; distal disinsertion of the long hallux flexor which was then positioned under the base of the first metatarsal and finally fixed on the distal dorsal segment of the metatarsophalangeal capsule; distal disinsertion of the anterior tibial tendon and tenodesis of the posterior tibial tendon. Weight bearing was allowed after pinning for one Month to position the axis of the first ray. Morphological results, recorded at 11 Years follow-up (mean) were satisfactory. There were no recurrent deformations and no residual instability of the first ray. The only observation was a minimal stiffness of the metatarsophalangeal joint with no tendency to degeneration.

Lano, a novel LAP protein directly connected to MAGUK proteins in epithelial cells.

Protein networks asymetrically distributed to basolateral and apical epithelial membranes maintain cell polarity and homeostasis of epithelial tissues. Genetic studies in non-vertebrates assigned two families of basolateral proteins, MAGUK (membrane-associated and guanylate kinase) and LAP (leucine-rich repeats and PDZ) proteins, to a common pathway crucial for the epithelial architecture and acting as a gatekeeper to malignancy. In mammals, three LAP proteins have been described, Densin-180, Erbin, and hScribble. Here, we identify a protein called Lano (LAP and no PDZ) only present in vertebrates and presenting strong identities with LAP proteins. Despite the lack of PDZ domain, Lano is located at the basolateral side of epithelial cells in a similar manner to Erbin and hScribble. Using in vitro and in vivo experiments, we demonstrate that Lano directly interacts with the PDZ domains of MAGUK proteins, including hDLG (human disc large), in epithelial cells. A second pool of Lano is complexed to Erbin. These LAP-MAGUK protein complexes coexist at the basolateral side of epithelial cells. We provide evidence for a direct interaction between LAP and MAGUK proteins, and we propose that various LAP-MAGUK networks targeted to the basolateral side of epithelial cells participate to homeostasis of epithelial tissues and tumor growth.

The carboxy-terminal sequence Asp427-Glu432 of beta-tubulin plays an important function in axonemal motility.

Flagellar motility is the result of specific interactions between axonemal microtubular proteins and the dynein motors. Tubulin, the main component of microtubule, is a very polymorphic protein resulting from the expression of several isogenes and from the existence of various post-translational modifications. In order to characterize tubulin isoforms and tubulin domains that are important for flagellar movement, we prepared monoclonal antibodies against axonemal proteins from whole sea-urchin sperm tails. The monoclonal antibodies obtained were screened for their potency to inhibit demembranated-reactivated sperm models and for their monospecific immunoreactivity on immunoblot. Among the different antibodies we obtained, D66 reacted specifically with a subset of beta-tubulin isoforms. Limited proteolysis, HPLC, peptide sequencing, mass spectroscopy and immunoblotting experiments indicated that D66 recognized an epitope localized in the primary sequence Gln423-Glu435 of the C-terminal domain of Lytechinus pictus beta2-tubulin, and that this sequence belongs to class IVb. The use of synthetic peptides and immunoblotting analysis further narrowed the amino acids important for antibody recognition to Asp427-Glu432. Because the primary effect of this antibody on sperm motility is to decrease the flagellar beat frequency, we suggest that this sequence is involved in the tubulin-dynein head interaction.

Tubulin polyglutamylase: partial purification and enzymatic properties.

In this work, we report on a novel enzyme, tubulin polyglutamylase, which catalyzes the posttranslational formation of polyglutamyl side chains onto alpha- and beta-tubulin. The length of the polyglutamyl side chain regulates the interaction between tubulin and various microtubule-associated proteins. We first developed an in vitro glutamylation assay. Activity measured in brain, a tissue particularly enriched with glutamylated tubulin, decreases during postnatal development. Thus, brains from 3-day-old mice were chosen as the starting material, and the enzyme was purified approximately 1000-fold. Its Mr was estimated to be 360K and its sedimentation coefficient 10 s. The enzyme catalyzes the MgATP-dependent addition of l-glutamate onto tubulin subunits. Microtubules are much better substrates than unpolymerized tubulin, and the reaction is very specific for glutamate, other amino acids or glutamate analogues not being substrates. Moreover, glutamyl units are added sequentially onto tubulin, leading to progressive elongation of the polyglutamyl side chains. Side chains of one to six or seven glutamyl units were obtained with microtubules, whereas much longer side chains (up to 15-20 units) were formed with unpolymerized tubulin. Interestingly, such very long polyglutamyl side chains were recently detected in some situations in vivo.